Recently, developments in the area of cloning and cloning procedures have expanded. For example, there have been several reports of live births of animals using cloning procedures. Live lambs were produced following nuclear transfer of cultured embryonic disc cells. Campbell et al. (1996) Nature 380:64-68. Campbell et al. disclose methods of transferring the nucleus of a quiescent cell into an enucleated oocyte to form a reconstructed embryo. The reconstructed embryo was developed until the morula to blastocyst stage in vivo prior to transfer into a recipient female.
Other reports of the live births of cloned mammals have also relied upon the transfer of a reconstructed embryo after it has reached the blastocyst stage of embryogenesis.
The present invention is based, in part, on the discovery that a reconstructed embryo which is transferred into a recipient mammal at the two to four cell stage of embryogenesis can develop into a cloned mammal. The mammal can be an embryo, a fetus, or a post natal mammal, e.g., an adult mammal.
Accordingly, in one aspect, the invention features a method of producing a non-human mammal, e.g., a cloned mammal, e.g., a goat, cow, pig, horse, sheep, llama, camel. The method includes maintaining a mammalian reconstructed embryo, e.g., a reconstructed embryo wherein the genome is derived from a somatic cell, in culture until the embryo is in the 2 to 8 cell stage, transferring the embryo at the 2 to 8 cell stage into a recipient mammal, and allowing the reconstructed embryo to develop into a mammal, to thereby produce a mammal.
In a preferred embodiment, the mammal develops from the reconstructed embryo. In another embodiment, the mammal is a descendant of a mammal which developed from the reconstructed embryo.
In a preferred embodiment, the reconstructed embryo is maintained in culture until the embryo is in the 2 to 8, the 2 to 6, the 2 to 4 cell stage of embryogenesis.
In a preferred embodiment, the genome of the reconstructed embryo is derived from: a somatic cell, e.g., a fibroblast or epithelial cell; a genetically engineered somatic cell, e.g., a somatic cell comprising a transgenic sequence.
In a preferred embodiment, the method further includes mating the mammal which develops from the reconstructed embryo with: a second mammal; a second mammal which develops from a reconstructed embryo or is descended from a mammal which developed from a reconstructed embryo; or a second mammal developed from a reconstructed embryo, or descended from a mammal which developed from a reconstructed embryo, which was formed from genetic material from the same animal, an animal of the same genotype, or same cell line, which supplied the genetic material for the first mammal. In a preferred embodiment, a first transgenic mammal which develops from the reconstructed embryo can be mated with a second transgenic mammal which developed from a reconstructed embryo and which contains a different transgene that the first transgenic mammal.
In a preferred embodiment, the mammal is a male mammal. In other preferred embodiments, the mammal is a female mammal. A female mammal can be induced to lactate and milk can be obtained from the mammal.
In a preferred embodiment: a product, e.g., a protein, e.g., a recombinant protein, e.g., a human protein, is recovered from the mammal; a product, e.g., a protein, e.g., a human protein, is recovered from the milk, urine, hair, blood, skin or meat of the mammal.
In a preferred embodiment, the mammal is: embryonic; fetal; or, postnatal, e.g., adult.
In a preferred embodiment, the genome of the reconstructed embryo is derived from a genetically engineered somatic cell, e.g., a transgenic cell or a cell which a nucleic acid has been introduced.
In another aspect, the invention features a method of producing a non-human mammal, e.g., a transgenic mammal, e.g., a goat, cow, pig, horse, sheep, llama, camel. The method includes maintaining a mammalian reconstructed embryo (e.g., a reconstructed embryo wherein its genome is derived from a genetically engineered somatic cell) in culture until the embryo is in the 2 to 8 cell stage, transferring the embryo at the 2 to 8 cell stage into a recipient mammal, and allowing the reconstructed embryo to develop into a mammal, to thereby produce a transgenic mammal.
In a preferred embodiment, the mammal develops from the reconstructed embryo. In another embodiment, the mammal is a descendant of a mammal which developed from the reconstructed embryo.
In a preferred embodiment, the reconstructed embryo is maintained in culture until the embryo is in the 2 to 8, the 2 to 6, the 2 to 4 cell stage of embryogenesis.
In a preferred embodiment, the method further includes mating the mammal which develops from the reconstructed embryo with: a second mammal; a second mammal which develops from a reconstructed embryo or is descended from a mammal which developed from a reconstructed embryo; or a second mammal developed from a reconstructed embryo, or descended from a mammal which developed from a reconstructed embryo, which was formed from genetic material from the same animal, an animal of the same genotype, or same cell line, which supplied the genetic material for the first mammal. In a preferred embodiment, a first transgenic mammal which develops from the reconstructed embryo can be mated with a second transgenic mammal which developed from a reconstructed embryo and which contains a different transgene that the first transgenic mammal.
In a preferred embodiment, the mammal is a male mammal. In other preferred embodiments, the mammal is a female mammal. A female mammal can be induced to lactate and milk can be obtained from the mammal.
In a preferred embodiment: a product, e.g., a protein, e.g., a recombinant protein, e.g., a human protein, is recovered from the mammal; a product, e.g., a protein, e.g., a human protein, is recovered from the milk, urine, hair, blood, skin or meat of the mammal.
In a preferred embodiment, the genome of the genetically engineered somatic cell includes a transgenic sequence. The transgenic sequence can be any of: a heterologous transgene, e.g., a human transgene; a knockout, knockin or other event which disrupts the expression of a mammalian gene; a sequence which encodes a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine promoter. The transgenic sequence can encode any product of interest such as a protein, polypeptide or peptide.
In a preferred embodiment, the transgenic sequence encodes any of: a hormone, an immunoglobulin, a plasma protein, and an enzyme. The transgenic sequence can encode any protein whose expression in the transgenic mammal is desired, e.g., any of: xcex1-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, fibrogen, glucocerebrosidase, glutamate decarboxylase, human serum albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythrpoietin, tissue plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and (xcex11-antitrypsin.
In a preferred embodiment, the transgenic sequence encodes a human protein.
In a preferred embodiment, the transgenic sequence is under the control of a promoter, e.g., a caprine or heterologous promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of: milk-specific promoters; blood-specific promoters; muscle-specific promoters; neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be, e.g., any of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a lactalbumin promoter.
In a preferred embodiment, a nucleic acid can be introduced into the genome of the genetically engineered somatic cell. The nucleic acid can be any of: a heterologous transgene, e.g., a human transgene; a knockout, knockin or other event which disrupts the expression of a mammalian gene; a sequence which encodes a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine or heterologous promoter. The nucleic acid sequence can encode any product of interest such as a protein, polypeptide or peptide.
In a preferred embodiment, the nucleic acid encodes any of: a hormone, an immunoglobulin, a plasma protein, and an enzyme. The nucleic acid sequence can encode any protein whose expression in the transgenic mammal is desired, e.g., any of: xcex1-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, fibrogen, glucocerebrosidase, glutamate decarboxylase, human serum albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythrpoietin, tissue plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and xcex11-antitrypsin.
In a preferred embodiment, the nucleic acid sequence encodes a human protein.
In a preferred embodiment, the nucleic acid sequence is under the control of a promoter, e.g., a caprine or heterologous promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of: milk-specific promoters; blood-specific promoters; muscle-specific promoters; neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be, e.g., any of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a lactalbumin promoter.
In another aspect, the invention features a method of producing a cloned goat. The method includes maintaining a caprine reconstructed embryo (e.g., a reconstructed embryo wherein its genome is derived from a caprine somatic cell) in culture until the embryo is in the 2 to 8 cell stage, transferring the embryo at the 2 to 8 cell stage into a recipient goat, and allowing the reconstructed embryo to develop into a goat, to thereby produce a goat.
In a preferred embodiment, the goat is: embryonic; fetal; or, postnatal, e.g., adult.
In a preferred embodiment, the goat develops from the reconstructed embryo. In another embodiment, the goat is a descendant of a goat which developed from the reconstructed embryo.
In a preferred embodiment, the reconstructed embryo is maintained in culture until the embryo is in the 2 to 8, the 2 to 6, the 2 to 4 cell stage of embryogenesis.
In a preferred embodiment, the genome of the reconstructed embryo is derived from: a caprine somatic cell, e.g., a fibroblast or epithelial cell; a genetically engineered caprine somatic cell, e.g., the genome of the caprine somatic cell comprises a transgenic sequence or a nucleic acid has been introduced into the genome of the somatic cell.
In a preferred embodiment, the method further includes mating the goat which develops from the reconstructed embryo with: a second goat; a second goat which develops from a reconstructed embryo or is descended from a goat which developed from a reconstructed embryo; or a second goat developed from a reconstructed embryo, or descended from a goat which developed from a reconstructed embryo, which was formed from genetic material from the same animal, an animal of the same genotype, or same cell line, which supplied the genetic material for the first goat. In a preferred embodiment, a first transgenic goat which develops from the reconstructed embryo can be mated with a second transgenic goat which developed from a reconstructed embryo and which contains a different transgene that the first transgenic goat.
In a preferred embodiment, the goat is a male goat. In other preferred embodiments, the goat is a female goat. A female goat can be induced to lactate and milk can be obtained from the goat.
In a preferred embodiment: a product, e.g., a protein, e.g., a recombinant protein, e.g., a human protein, is recovered from the goat; a product, e.g., a protein, e.g., a human protein, is recovered from the milk, urine, hair, blood, skin or meat of the goat.
In another aspect, the invention features a method of producing a transgenic goat. The method includes maintaining a caprine reconstructed embryo (e.g., a reconstructed embryo wherein its genome is derived from a genetically engineered somatic cell) in culture until the embryo is in the 2 to 8 cell stage, transferring the embryo at the 2 to 8 a cell stage into a recipient goat, and allowing the reconstructed embryo to develop into a goat, to thereby produce a transgenic goat.
In a preferred embodiment, the goat is: embryonic; fetal; or, postnatal, e.g., adult.
In a preferred embodiment, the goat develops from the reconstructed embryo. In another embodiment, the goat is a descendant of a goat which developed from the reconstructed embryo.
In a preferred embodiment, the reconstructed embryo is maintained in culture until the embryo is in the 2 to 8, the 2 to 6, the 2 to 4 cell stage of embryogenesis.
In a preferred embodiment, the genome of the reconstructed embryo is derived from a somatic cell, e.g., a fibroblast or epithelial cell.
In a preferred embodiment, the method further includes mating the goat which develops from the reconstructed embryo with: a second goat; a second goat which develops from a reconstructed embryo or is descended from a goat which developed from a reconstructed embryo; or a second goat developed from a reconstructed embryo, or descended from a goat which developed from a reconstructed embryo, which was formed from genetic material from the same animal, an animal of the same genotype, or same cell line, which supplied the genetic material for the first goat. In a preferred embodiment, a first transgenic goat which develops from the reconstructed embryo can be mated with a second transgenic goat which developed from a reconstructed embryo and which contains a different transgene that the first transgenic goat.
In a preferred embodiment, the goat is a male goat. In other preferred embodiments, the goat is a female goat. A female goat can be induced to lactate and milk can be obtained from the goat.
In a preferred embodiment: a product, e.g., a protein, e.g., a recombinant protein, e.g., a human protein, is recovered from the goat; a product, e.g., a protein, e.g., a human protein, is recovered from the milk, urine, hair, blood, skin or meat of the goat.
In a preferred embodiment, the genome of the genetically engineered somatic cell includes a transgenic sequence. The transgenic sequence can be any of: a heterologous transgene, e.g., a human transgene; a knockout, knockin or other event which disrupts the expression of a mammalian gene; a sequence which encodes a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine or heterologous promoter. The transgenic sequence can encode any product of interest such as a protein, polypeptide or peptide.
In a preferred embodiment, the transgenic sequence encodes any of: a hormone, an immunoglobulin, a plasma protein, and an enzyme. The transgenic sequence can encode any protein whose expression in the transgenic mammal is desired, e.g., any of: xcex1-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, fibrogen, glucocerebrosidase, glutamate decarboxylase, human serum albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythrpoietin, tissue plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and xcex11-antitrypsin.
In a preferred embodiment, the transgenic sequence encodes a human protein.
In a preferred embodiment, the transgenic sequence is under the control of a promoter, e.g., a caprine or heterologous promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of: milk-specific promoters; blood-specific promoters; muscle-specific promoters; neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be, e.g., any of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a lactalbumin promoter.
In a preferred embodiment, a nucleic acid has been introduced into the genome of the genetically engineered somatic cell. The nucleic acid sequence can be any of: a heterologous transgene, e.g., a human transgene; a knockout, knockin or other event which disrupts the expression of a mammalian gene; a sequence which encodes a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine or heterologous promoter. The transgenic sequence can encode any product of interest such as a protein, polypeptide or peptide.
In a preferred embodiment, the nucleic acid encodes any of: a hormone, an immunoglobulin, a plasma protein, and an enzyme. The nucleic acid sequence can encode any protein whose expression in the transgenic mammal is desired, e.g., any of: xcex1-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, fibrogen, glucocerebrosidase, glutamate decarboxylase, human serum albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythrpoietin, tissue plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and xcex11-antitrypsin.
In a preferred embodiment, the nucleic acid sequence encodes a human protein.
In a preferred embodiment, the nucleic acid sequence is under the control of a promoter, e.g., a caprine or heterologous promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of: milk-specific promoters; blood-specific promoters; muscle-specific promoters; neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be, e.g., any of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a lactalbumin promoter.
In another aspect, the invention features a kit. The kit includes a reconstructed embryo which is in the 2 to 8 cell stage. In a preferred embodiment, the kit further includes instructions for producing a mammal, e.g., an embryonic, fetal or postnatal mammal.
In another aspect, the invention features a kit which includes a later stage embryo, e.g., an embryo after the 8 cell stage, or a fetus, obtained, e.g., by the methods described herein.
The terms protein, polypeptide and peptide are used interchangeably herein.
The phrase xe2x80x9ca genome derived from a somatic cellxe2x80x9d, as used herein, refers to the nuclear transfer of a somatic cell, e.g., the chromosomal genome of a somatic cell, into a functionally enucleated oocyte.
The term xe2x80x9cgenetically engineeredxe2x80x9d, as used herein, refers to a cell altered by human intervention, e.g., a transgenic cell or other cell into which a nucleic acid has been introduced.
As used herein, the term xe2x80x9ctransgenic sequencexe2x80x9d refers to a nucleic acid sequence (e.g., encoding one or more human proteins), which is inserted by artifice into a cell. The transgenic sequence, also referred to herein as a transgene, can become part of the genome of an animal which develops in whole or in part from that cell. In embodiments of the invention, the transgenic sequence is integrated into the chromosomal genome. If the transgenic sequence is integrated into the genome it results, merely by virtue of its insertion, in a change in the nucleic acid sequence of the genome into which it is inserted. A transgenic sequence can be partly or entirely species-heterologous, i.e., the transgenic sequence, or a portion thereof, can be from a species which is different from the cell into which it is introduced. A transgenic sequence can be partly or entirely species-homologous, i.e., the transgenic sequence, or a portion thereof, can be from the same species as is the cell into which it is introduced. If a transgenic sequence is homologous (in the sequence sense or in the species-homologous sense) to an endogenous gene of the cell into which it is introduced, then the transgenic sequence, preferably, has one or more of the following characteristics: it is designed for insertion, or is inserted, into the cell""s genome in such a way as to alter the sequence of the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the endogenous gene or its insertion results in a change in the sequence of the endogenous endogenous gene); it includes a mutation, e.g., a mutation which results in misexpression of the transgenic sequence; by virtue of its insertion, it can result in misexpression of the gene into which it is inserted, e.g., the insertion can result in a knockout of the gene into which it is inserted. A transgenic sequence can include one or more transcriptional regulatory sequences and any other nucleic acid sequences, such as introns, that may be necessary for a desired level or pattern of expression of a selected nucleic acid, all operably linked to the selected nucleic acid. The transgenic sequence can include an enhancer sequence and or sequences which allow for secretion.
The term xe2x80x9cheterologous promoterxe2x80x9d as used herein, refers to a promoter which is not normally associated with the gene it controls or which is heterologous to the mammal into which it is introduced.
The terms xe2x80x9creconstructed embryoxe2x80x9d, xe2x80x9creconstituted embryoxe2x80x9d and xe2x80x9cnuclear transfer unitxe2x80x9d are used interchangeably herein.
Other features and advantages of the invention will be apparent from the following description and from the claims.
Donor Cells
Somatic cells can supply the genome for producing a reconstructed embryo in the methods described herein. The term xe2x80x9csomatic cellxe2x80x9d, as used herein, refers to a differentiated cell. The cell can be a somatic cell or a cell that is committed to a somatic cell lineage. Alternatively, any of the methods and animals described herein can utilize a diploid stem cell that gives rise to a germ cell in order to supply the genome for producing a reconstructed embryo.
The somatic cell can be from an animal or from a cell culture. If taken from an animal, the animal can be at any stage of development, e.g., an embryo, a fetus or an adult. Embryonic cells are preferred. Embryonic cells can include embryonic stem cells as well as embryonic cells committed to a somatic cell lineage. Such cells can be obtained from the endoderm, mesoderm or ectoderm of the embryo. Preferably, the embryonic cells are committed to somatic cell lineage. Embryonic cells committed to a somatic cell lineage refer to cells isolated on or after day 10 of embryogenesis. However, cells can be obtained prior to day ten of embryogenesis. If a cell line is used as a source of a chromosomal genome, primary cells are preferred. The term xe2x80x9cprimary cell linexe2x80x9d as used herein includes primary cell lines as well as primary-derived cell lines.
Suitable somatic cells include fibroblasts (e.g., primary fibroblasts, e.g., embryonic primary fibroblasts), muscle cells (e.g., myocytes), cumulus cells, neural cells, and mammary cells. Other suitable cells include hepatocytes and pancreatic islets. Preferably, the somatic cell is an embryonic somatic cell, e.g., a cell isolated on or after day 10 of embryogenesis. The genome of the somatic cells can be the naturally occurring genome, e.g., for the production of cloned mammals, or the genome can be genetically altered to comprise a transgenic sequence, e.g., for the production of transgenic cloned mammals.
Somatic cells can be obtained by, for example, dissociation of tissue, e.g., by mechanical (e.g., chopping, mincing) or enzymatic means (e.g., trypsinization) to obtain a cell suspension and then by culturing the cells until a confluent monolayer is obtained. The somatic cells can then be harvested and prepared for cryopreservation, or maintained as a stock culture. The isolation of caprine somatic cells, e.g., fibroblasts, is described herein.
The somatic cell can be a quiescent or non-quiescent somatic cell. xe2x80x9cNon-quiescentxe2x80x9d, as used herein, refers to a cell in mitotic cell cycle. The mitotic cell cycle has four distinct phases, G1, S, G2 and M. The beginning event in the cell cycle, called START, takes place during the G1 phase. xe2x80x9cSTARTxe2x80x9d as used herein refers to early G1 stage of the cell cycle prior to the commitment of a cell to proceeding through the cell cycle. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 11 hours after a cell enters the G1 stage, the cell is considered prior to START. The decision as to whether the cell will undergo another cell cycle is made at START. Once the cell has passed through START, it passes through the remainder of the G1 phase (i.e., the pre-DNA synthesis stage). The S phase is the DNA synthesis stage, which is followed by the G2 phase, the stage between synthesis and mitosis. Mitosis takes place during the M phase. If at START, the cell does not undergo another cell cycle, the cell becomes quiescent. In addition, a cell can be induced to exit the cell cycle and become quiescent. A xe2x80x9cquiescentxe2x80x9d cell, also referred to as a cell in G0 phase, refers to a cell which is not in any of the four phases of the cell cycle. Preferably, the somatic cell is a cell in the G0 phase or the G1 phase of the mitotic cell cycle.
Using donor somatic cells at certain phases of the cell cycle, e.g., G0 or G1 phase, can allow for synchronization between the oocyte and the genome of the somatic cell. For example, reconstruction of an oocyte in metaphase II by introduction of a nucleus of a somatic cell in G0 or G1, e.g., by simultaneous activation and fusion, can mimic the events occurring during fertilization.
Methods of determining which phase of the cell cycle a cell is in are known. For example, as described below in the Examples, various markers are present at different stages of the cell cycle. Such markers can include cyclins D 1, 2, 3 and proliferating cell nuclear antigen (PCNA) for G1, and BrDu to detect DNA synthetic activity. In addition, cells can be induced to enter the G0 stage by culturing the cells on serum-deprived medium. Alternatively, cells in G0 stage can be induced to enter the cell cycle, i.e., at G1 stage, by serum activation.
Sources of Genetically Engineered Somatic Cells
Transgenic Mammals
Methods for generating non-human transgenic mammals which can be used as a source of somatic cells in the invention are known in the art. Such methods can involve introducing DNA constructs into the germ line of a mammal to make a transgenic mammal. For example, one or several copies of the construct may be incorporated into the genome of a mammalian embryo by standard transgenic techniques.
Although goats are a preferred source of genetically engineered somatic cells, other non-human mammals can be used. Preferred non-human mammals are ruminants, e.g., cows, sheep, camels or goats. Goats of Swiss origin, e.g., the Alpine, Saanen and Toggenburg breed goats, are useful in the methods described herein. Additional examples of preferred non-human animals include oxen, horses, llamas, and pigs. The mammal used as the source of genetically engineered cells will depend on the transgenic mammal to be obtained by the methods of the invention as, by way of example, a goat genome should be introduced into a goat functionally enucleated oocyte.
Preferably, the somatic cells for use in the invention are obtained from a transgenic goat. Methods of producing transgenic goats are known in the art. For example, a transgene can be introduced into the germline of a goat by microinjection as described, for example, in Ebert et al. (1994) Bio/Technology 12:699, hereby incorporated by reference.
Other transgenic non-human animals to be used as a source of genetically engineered somatic cells can be produced by introducing a transgene into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness. In addition, the haplotype is a significant factor.
Transfected Cell Lines
Genetically engineered somatic cells for use in the invention can be obtained from a cell line into which a nucleic acid of interest, e.g., a nucleic acid which encodes a protein, has been introduced.
A construct can be introduced into a cell via conventional transformation or transfection techniques. As used herein, the terms xe2x80x9ctransfectionxe2x80x9d and xe2x80x9ctransformationxe2x80x9d include a variety of techniques for introducing a transgenic sequence into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextrane-mediated transfection, lipofection, or electroporation. In addition, biological vectors, e.g., viral vectors can be used as described below. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al., Molecular Cloning: A Laboratory Manuel, 2nd ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other suitable laboratory manuals.
Two useful approaches are electroporation and lipofection. Brief examples of each are described below.
The DNA construct can be stably introduced into a donor somatic cell line by electroporation using the following protocol: somatic cells, e.g., fibroblasts, e.g., embryonic fibroblasts, are resuspended in PBS at about 4xc3x97106 cells/ml. Fifty micorgrams of linearized DNA is added to the 0.5 ml cell suspension, and the suspension is placed in a 0.4 cm electrode gap cuvette (Biorad). Electroporation is performed using a Biorad Gene Pulser electroporator with a 330 volt pulse at 25 mA, 1000 microFarad and infinite resistance. If the DNA construct contains a Neomyocin resistance gene for selection, neomyocin resistant clones are selected following incubation with 350 microgram/ml of G418 (GibcoBRL) for 15 days.
The DNA construct can be stably introduced into a donor somatic cell line by lipofection using a protocol such as the following: about 2xc3x97105 cells are plated into a 3.5 cmiameter well and transfected with 2 micrograms of linearized DNA using LipfectAMINE(trademark) (GibcoBRL). Forty-eight hours after transfection, the cells are split 1:1000 and 1:5000 and, if the DNA construct contains a neomyosin resistance gene for selection, G418 is added to a final concentration of 0.35 mg/ml. Neomyocin resistant clones are isolated and expanded for cyropreservation as well as nuclear transfer.
Tissue-Specific Expression of Proteins
It is often desirable to express a protein, e.g., a heterologous protein, in a specific tissue or fluid, e.g., the milk, of a transgenic animal. The heterologous protein can be recovered from the tissue or fluid in which it is expressed. For example, it is often desirable to express the heterologous protein in milk. Methods for producing a heterologous protein under the control of a milk specific promoter are described below. In addition, other tissue-specific promoters, as well as, other regulatory elements, e.g., signal sequences and sequence which enhance secretion of non-secreted proteins, are described below.
Milk Specific Promoters
Useful transcriptional promoters are those promoters that are preferentially activated in mammary epithelial cells, including promoters that control the genes encoding milk proteins such as caseins, beta lactoglobulin (Clark et al., (1989) Bio/Technology 7: 487-492), whey acid protein (Gordon et al. (1987) Bio/Technology 5: 1183-1187), and lactalbumin (Soulier et al., (1992) FEBS Letts. 297: 13). Casein promoters may be derived from the alpha, beta, gamma or kappa casein genes of any mammalian species; a preferred promoter is derived from the goat beta casein gene (DiTullio, (1992) Bio/Technology 10:74-77). Milk-specific protein promoter or the promoters that are specifically activated in mammary tissue can be derived from cDNA or genomic sequences. Preferably, they are genomic in origin.
DNA sequence information is available for the mammary gland specific genes listed above, in at least one, and often in several organisms. See, e.g., Richards et al., J. Biol. Chem. 256, 526-532 (1981) (xcex1-lactalbumin rat); Campbell et al., Nucleic Acids Res. 12, 8685-8697 (1984) (rat WAP); Jones et al., J. Biol. Chem. 260, 7042-7050 (1985) (rat xcex2-casein); Yu-Lee and Rosen, J. Biol. Chem. 258, 10794-10804 (1983) (rat xcex3-casein); Hall, Biochem. J. 242, 735-742 (1987) (xcex1-lactalbumin human); Stewart, Nucleic Acids Res. 12, 389 (1984) (bovine xcex1s1 and xcexa casein cDNAs); Gorodetsky et al., Gene 66, 87-96 (1988) (bovine xcex2 casein); Alexander et al., Eur. J. Biochem. 178, 395-401 (1988) (bovine xcexa casein); Brignon et al., FEBS Lett. 188, 48-55 (1977) (bovine xcex1S2 casein); Jamieson et al., Gene 61, 85-90 (1987), Ivanov et al., Biol. Chem. Hoppe-Seyler 369, 425-429 (1988), Alexander et al., Nucleic Acids Res. 17, 6739 (1989) (bovine xcex2 lactoglobulin); Vilotte et al., Biochimie 69, 609-620 (1987) (bovine xcex1-lactalbumin). The structure and function of the various milk protein genes are reviewed by Mercier and Vilotte, J. Dairy Sci. 76, 3079-3098 (1993) (incorporated by reference in its entirety for all purposes). If additional flanking sequence are useful in optimizing expression of the heterologous protein, such sequences can be cloned using the existing sequences as probes. Mammary-gland specific regulatory sequences from different organisms can be obtained by screening libraries from such organisms using known cognate nucleotide sequences, or antibodies to cognate proteins as probes.
Signal Sequences
Useful signal sequences are milk-specific signal sequences or other signal sequences which result in the secretion of eukaryotic or prokaryotic proteins. Preferably, the signal sequence is selected from milk-specific signal sequences, i.e., it is from a gene which encodes a product secreted into milk. Most preferably, the milk-specific signal sequence is related to the milk-specific promoter used in the construct, which are described below. The size of the signal sequence is not critical. All that is required is that the sequence be of a sufficient size to effect secretion of the desired recombinant protein, e.g., in the mammary tissue. For example, signal sequences from genes coding for caseins, e.g., alpha, beta, gamma or kappa caseins, beta lactoglobulin, whey acid protein, and lactalbumin can be used. A preferred signal sequence is the goat xcex2-casein signal sequence.
Signal sequences from other secreted proteins, e.g., proteins secreted by kidney cells, pancreatic cells or liver cells, can also be used. Preferably, the signal sequence results in the secretion of proteins into, for example, urine or blood.
Amino-Terminal Regions of Secreted Proteins
A non-secreted protein can also be modified in such a manner that it is secreted such as by inclusion in the protein to be secreted of all or part of the coding sequence of a protein which is normally secreted. Preferably the entire sequence of the protein which is normally secreted is not included in the sequence of the protein but rather only a sufficient portion of the amino terminal end of the protein which is normally secreted to result in secretion of the protein. For example, a protein which is not normally secreted is fused (usually at its amino terminal end) to an amino terminal portion of a protein which is normally secreted.
In one aspect, the protein which is normally secreted is a protein which is normally secreted in milk. Such proteins include proteins secreted by mammary epithelial cells, milk proteins such as caseins, beta lactoglobulin, whey acid protein, and lactalbumin. Casein proteins include alpha, beta, gamma or kappa casein genes of any mammalian species. A preferred protein is beta casein, e.g., goat beta casein. The sequences which encode the secreted protein can be derived from either cDNA or genomic sequences. Preferably, they are genomic in origin, and include one or more introns.
Other Tissue-Specific Promoters
Other tissue-specific promoters which provide expression in a particular tissue can be used. Tissue specific promoters are promoters which are expressed more strongly in a particular tissue than in others. Tissue specific promoters are often expressed essentially exclusively in the specific tissue.
Tissue-specific promoters which can be used include: a neural-specific promoter, e.g., nestin, Wnt-1, Pax-1, Engrailed-1, Engrailed-2, Sonic hedgehog; a liver-specific promoter, e.g., albumin, alpha-1 antirypsin; a muscle-specific promoter, e.g., myogenin, actin, MyoD, myosin; an oocyte specific promoter, e.g., ZP1, ZP2, ZP3; a testes-specific promoter, e.g., protamin, fertilin, synaptonemal complex protein-1; a blood-specific promoter, e.g., globulin, GATA-1, porphobilinogen deaminase; a lung-specific promoter, e.g., surfactant protein C; a skin- or wool-specific promoter, e.g., keratin, elastin; endothelium-specific promoters, e.g., Tie-1, Tie-2; and a bone-specific promoter, e.g., BMP.
In addition, general promoters can be used for expression in several tissues. Examples of general promoters include xcex2-actin, ROSA-21, PGK, FOS, c-myc, Jun-A, and Jun-B.
DNA Constructs
A cassette which encodes a heterologous protein can be assembled as a construct which includes a promoter for a specific tissue, e.g., for mammary epithelial cells, e.g., a casein promoter, e.g., a goat beta casein promoter, a milk-specific signal sequence, e.g., a casein signal sequence, e.g., a xcex2-casein signal sequence, and a DNA encoding the heterologous protein.
The construct can also include a 3xe2x80x2 untranslated region downstream of the DNA sequence coding for the non-secreted protein. Such regions can stabilize the RNA transcript of the expression system and thus increases the yield of desired protein from the expression system. Among the 3xe2x80x2 untranslated regions useful in the constructs for use in the invention are sequences that provide a poly A signal. Such sequences may be derived, e.g., from the SV40 small t antigen, the casein 3xe2x80x2 untranslated region or other 3xe2x80x2 untranslated sequences well known in the art. In one aspect, the 3xe2x80x2 untranslated region is derived from a milk specific protein. The length of the 3xe2x80x2 untranslated region is not critical but the stabilizing effect of its poly A transcript appears important in stabilizing the RNA of the expression sequence.
Optionally, the construct can include a 5xe2x80x2 untranslated region between the promoter and the DNA sequence encoding the signal sequence. Such untranslated regions can be from the same control region from which promoter is taken or can be from a different gene, e.g., they may be derived from other synthetic, semi-synthetic or natural sources. Again their specific length is not critical, however, they appear to be useful in improving the level of expression.
The construct can also include about 10%, 20%, 30%, or more of the N-terminal coding region of a gene preferentially expressed in mammary epithelial cells. For example, the N-terminal coding region can correspond to the promoter used, e.g., a goat xcex2-casein N-terminal coding region.
The construct can be prepared using methods known in the art. The construct can be prepared as part of a larger plasmid. Such preparation allows the cloning and selection of the correct constructions in an efficient manner. The construct can be located between convenient restriction sites on the plasmid so that they can be easily isolated from the remaining plasmid sequences for incorporation into the desired mammal.
Heterologous Proteins
Transgenic sequences encoding heterologous proteins can be introduced into the germline of a non-human mammal or can be transfected into a cell line to provide a source of genetically engineered somatic cells as described above. The protein can be a complex or multimeric protein, e.g., a homo- or heteromultimer, e.g., proteins which naturally occur as homo- or heteromultimers, e.g., homo- or hetero-dimers, trimers or tetramers. The protein can be a protein which is processed by removal, e.g., cleavage, of N-terminus, C-terminus or internal fragments. Even complex proteins can be expressed in active form. Protein encoding sequences which can be introduced into the genome of mammal, e.g., goats, include glycoproteins, neuropeptides, immunoglobulins, enzymes, peptides and hormones. The protein may be a naturally occurring protein or a recombinant protein, e.g., a fragment, fusion protein, e.g., an immunoglogulin fusion protein, or mutien. It may be human or non-human in origin. The heterologous protein may be a potential therapeutic or pharmaceutical agent such as, but not limited to: alpha-1 proteinase inhibitor, alpha-1 antitrypsine, alkaline phosphatase, angiogenin, antithrombin III, any of the blood clotting factors including Factor VIII, Factor IX, and Factor X chitinase, erythropoietin, extracellular superoxide dismutase, fibrinogen, glucocerebrosidase, glutamate decarboxylase, human growth factor, human serum albumin, immunoglobulin, insulin, myelin basic protein, proinsulin, prolactin, soluble CD4 or a component or complex thereof, lactoferrin, lactoglobulin, lysozyme, lactalbumin, tissue plasminogen activator or a variant thereof.
Immunoglobulins are particularly preferred heterologous protiens. Examples of immunoglobulins include IgA, IgG, IgE, IgM, chimeric antibodies, humanized antibodies, recombinant antibodies, single chain antibodies and antibody-protein fusions.
Nucleotide sequence information is available for several of the genes encoding the heterologous proteins listed above, in at least one, and often in several organisms. See e.g., Long et al. (1984) Biochem. 23(21):4828-4837 (aplha-1 antitrypsin); Mitchell et al. (1986) Prot. Natl. Acad. Sci USA 83:7182-7186 (alkaline phosphatase); Schneider et al. (1988) EMBO J. 7(13):4151-4156 (angiogenin); Bock et al. (1988) Biochem. 27(16):6171-6178 (antithrombin III); Olds et al. (1991) Br. J. Haematol. 78(3):408-413 (antithrombin III); Lin et al. (1985) Proc. Natl. Acad. Sci. USA 82(22):7580-7584 (erythropoeitin); U.S. Pat. No. 5,614,184 (erythropoietin); Horowitz et al. (1989) Genomics 4(1):87-96 (glucocerebrosidase); Kelly et al. (1992) Ann. Hum. Genet. 56(3):255-265 (glutamte decarboxylase); U.S. Pat. No. 5,707,828 (human serum albumin); U.S. Pat. No. 5,652,352 (human serum albumin); Lawn et al. (1981) Nucleic Acid Res. 9(22):6103-6114 (human serum albumin); Kamholz et al. (1986) Prot. Natl. Acad. Sci. USA 83(13):4962-4966 (myelin basic protein); Hiraoka et al. (1991) Mol. Cell Endocrinol. 75(1):71-80 (prolactin); U.S. Pat. No. 5,571,896 (lactoferrin); Pennica et al. (1983) Nature 301(5897):214-221 (tissue plasminogen activator); Sarafanov et al. (1995) Mol. Biol. 29:161-165, the contents of which are incorporated herein by reference.
Oocytes
Suitable sources of oocytes include goat, cow, sheep, horse, pig, llama, camel, etc. Preferably the oocyte is obtained from a goat. Oocytes for use in the invention include oocytes in metaphase II stage, e.g., oocytes arrested in metaphase II, and telophase II. Oocytes in metaphase II contain one polar body, whereas oocytes in telophase can be identified based on the presence of a protrusion of the plasma membrane from the second polar body up to the formation of a second polar body. In addition, oocytes in metaphase II can be distinguished from oocytes in telophase II based on biochemical and/or developmental distinctions. For example, oocytes in metaphase II can be in an arrested state, whereas oocytes in telophase are in an activated state.
Occytes can be obtained at various times during a goat""s reproductive cycle. For example, at given times during the reproductive cycle, a significant percentage of the oocytes, e.g., about 55%, 60%, 65%, 70%, 75%, 80% or more, are oocytes in telophase. In addition, oocytes at various stages of the cell cycle can be obtained and then induced in vitro to enter a particular stage of meiosis. For example, oocytes cultured on serum-starved medium become arrested in metaphase. In addition, arrested oocytes can be induced to enter telophase by serum activation. Thus, oocytes in telophase can be easily obtained for use in the invention. Thus, oocytes can be matured in vitro before they are used to form a reconstructed embryo. This process usually requires collecting immature oocytes from mammalian ovaries, e.g., a caprine ovary, and maturing the oocyte in a medium prior to enucleation until the oocyte reaches the desired meiotic stage, e.g., metaphase or telophase. In addition, oocytes that have been matured in vivo can be used to form a reconstructed embryo.
Oocytes can be collected from a female mammal during superovulation. Briefly, oocytes, e.g., caprine oocytes, can be recovered surgically by flushing the oocytes from the oviduct of the female donor. Methods of inducing superovulation in goats and the collection of caprine oocytes is described herein.
Preferably, the mitotic stage of the oocyte, e.g., metaphase II or telophase II, correlates to the stage of the cell cycle of the donor somatic cell. The correlation between the meiotic stage of the oocyte and the mitotic stage of the cell cycle of the donor somatic cell is referred to herein as xe2x80x9csynchronizationxe2x80x9d. For example, reconstruction of an oocyte in metaphase II by introduction of a nucleus of a somatic cell in G0 or G1, e.g., by simultaneous activation and fusion, can mimic the events occurring during fertilization. By way of another example, an oocyte in telophase fused, e.g., by simultaneous activation and fusion, with the genome of a somatic cell in G1 prior to START, provides a synchronization between the oocyte and the donor nuclei.
Functional Enucleation
The donor oocyte, e.g., caprine oocyte, should be functionally enucleated such that the endogenous genome of the oocyte is incapable of functioning, e.g., replicating or synthesizing DNA. Methods of functionally enucleating an oocyte include: removing the genome from the oocyte (i.e., enucleation); inactivating DNA within the oocyte, e.g., by irradiation (e.g., by X-ray irradiation, or laser irradiation); chemical inactivation, or the like.
Enucleation
One method of rendering the genome of an oocyte incapable of functioning is to remove the genome from the oocyte (i.e., enucleation). A micropipette or needle can be inserted into the zona pellicuda in order to remove nuclear material from an oocyte. For example, metaphase II stage oocytes which have one polar body can be enucleated with a micropipette by aspirating the first polar body and adjacent cytoplasm surrounding the polar body, e.g., approximately 20%, 30%, 40%, 50%, 60% of the cytoplasm, which presumably contains the metaphase plate. Telphase stage oocytes which have two polar bodies can be enucleated with a micropipette or needle by removing the second polar body and surrounding cytoplasm, e.g., approximately 5%, 10%, 20%, 30%, 40%, 50%, 60% of cytoplasm. Specifically, oocytes in telophase stage can be enucleated at any point from the presence of a protrusion in the plasma membrane from the second polar body up to the formation of the second polar body. Thus, as used herein, oocytes which demonstrate a protrusion in the plasma membrane, usually with a spindle abutted to it, up to extrusion of the second polar body are considered to be oocytes in telophase. Alternatively, oocytes which have one clear and distinct polar body with no evidence of protrusion are considered to be oocytes in metaphase. Methods of enucleating an oocyte, e.g., a caprine oocyte, are described in further detail in the Examples.
Irradiation
The oocyte can be functionally enucleated by inactivating the endogenous DNA of the oocyte using irradiation. Methods of using irradiation are known in the art and described, for example, in Bradshaw et al. (1995) Molecul. Reprod. Dev. 41:503-512, the contents of which is incorporated herein by reference.
Chemical Inactivation
The oocyte can be functionally enucleated by chemically inactivating the endogenous DNA of the oocyte. Methods of chemically inactivating the DNA are known in the art. For example, chemical inactivation can be performed using the etopsoide-cycloheximide method as described in Fulkaj and Moore (1993) Molecul. Reprod. Dev. 34:427-430, the content of which are incorporated herein by reference.
Introduction of a Functional Chromosomal Genome Into an Oocyte
Methods described herein can include the introduction of a functional chromosomal genome into an oocyte, e.g., a functionally enucleated oocyte, e.g., an enucleated oocyte, to form a reconstructed embryo. The functional chromosomal genome directs the development of a cloned or transgenic animal which arises from the reconstructed embryo. Methods which result in the transfer of an essentially intact chromosomal genome to the oocyte can be used. Examples include fusion of a cell which contains the functional chromosomal genome with the oocyte and nuclear injection, i.e., direct transfer of the nucleus into the oocyte.
Fusion
Fusion of the somatic cell with an oocyte can be performed by, for example, electrofusion, viral fusion, biochemical reagent fusion (e.g., HA protein), or chemical fusion (e.g., with polyethylene glycol (PEG) or ethanol).
Fusion of the somatic cell with the oocyte and activation can be performed simultaneously. For example, the nucleus of the somatic cell can be deposited within the zona pelliduca which contains the oocyte. The steps of fusing the nucleus with the oocyte and activation can then be performed simultaneously by, for example, applying an electric field. Methods of simultaneous fusion and activation of a somatic cell and an oocyte are described herein.
Activation of a Recombinant Embryo
Activation refers to the beginning of embryonic development, e.g., replication and DNA synthesis. Activation can be induced by, for example, electric shock (e.g., in electrofusion), the use of ionophores, ethanol activation, or the oocyte can be obtained during a stage in which it is naturally activated, e.g., an oocyte in telophase.
Electrofusion
A reconstructed embryo can be activated using electric shock, i.e., electrofusion. The use of electrofusion allows for the fusion of the somatic cell with the oocyte and activation to be performed simultaneously.
Chambers, such as the BTX 200 Embryomanipulation System, for carrying out electrofusion are commercially available from, for example, BTX, San Diego. Methods for performing electrofusion to fuse a somatic cell, e.g., a caprine somatic cell, and an oocyte, e.g., an enucleated oocyte, e.g., an enucleated caprine oocyte, are described herein.
Ionophores
In addition, the reconstructed embryo can be activated by ionophore activation. Using an ionophore, e.g., a calcium ionophore, the calcium concentration across the membrane of the reconstructed embryo is changed. As the free calcium concentration in the cell increases, there is a decrease in phosphorylation of intracellular proteins and the oocyte is activated. Such methods of activation are described, for example, in U.S. Pat. No. 5,496,720, the contents of which are incorporated by reference.
Ethanol Activation
Prior to enucleation, an oocyte, e.g., an oocyte in metaphase II, can be activated with ethanol according to the ethanol activation treatment as described in Presicce and Yang (1994) Mol. Reprod. Dev. 37:61-68, and Bordignon and Smith (1998) Mol. Reprod. Dev. 49:29-36, the contents of which are incorporated herein by reference.
Ooctyes in Telophase
Oocytes in telophase are generally already activated. Thus, these cells often naturally exhibit a decrease in calcium concentration which prevents fertilization and allows the embryo to develop.
Transfer of Reconstructed Embryos
A reconstructed embryo of the invention can be transferred to a recipient doe and allowed to develop into a cloned or transgenic mammal, e.g., a cloned or transgenic goat. For example, the reconstructed embryo can be transferred via the fimbria into the oviductal lumen of each recipient doe as described below in the Examples. In addition, methods of transferring an embryo to a recipient mammal are known in the art and described, for example, in Ebert et al. (1994) Bio/Technology 12:699.
The reconstructed embryo can be maintained in a culture until at least first cleavage (2-cell stage) up to the eight cell-stage of embryogenesis, preferably the embryos are transferred at 2-cell or 4 cell-stage. Various culture media for embryo development are known in the art. For example, the reconstructed embryo can be co-cultured with oviductal epithelial cell monolayer derived from the type of mammal to be provided by the invention. Methods of obtaining goat oviductal epithelial cells (GOEC), maintaining the cells in a co-culture are described in the Examples below.